Introduction

Molecular biologists and genetic engineers work with such small quantities that special tools had to be developed to ensure accurate measurements. One of these tools is the digital micropipet. For biotechnology, it is as common a tool as a graduated cylinder. Without such precision, genetic engineering would be severely hampered.

Objectives

* The student will correctly use the micropipet.

* The student will mix liquids in a microcentrifuge tube.

* The student will load a gel.

* The student will convert measurements between the units microliters (ul) and milliliters (ml).

Materials

1. Digital micropipet 5. Tubes of H2O and loading dye

2. Box of micropipet tips 6. An agar gel (2%)

3. Tubes of food coloring 7. Parafilm

4. Waste container 8. Large culture bowl

Procedure

Part A: Measurement

You already know how to measure volume in milliliters (ml). This investigation will teach you how to use the micropipet so that you will be able to carry out future labs involving more critical measurements using a special unit called the microliter (ul). Below are some equivalents for these units. Fill in the blanks below with the correct values.

1ml =1/1000 liter _____ml = 1 liter

1ul = 1,000,000 liter _____ul = 1 liter

1,000 ul =1ml _____ml = 1 ul

Part B: Some DON'TS

1. Do not use the micropipet without a disposable tip in place. Moisture may be drawn up inside the pipettor damaging the internal piston.

2. Do not lay either a liquid laden micropipet or a pipettor with an empty wet tip down. Moisture can run back inside causing damage to the piston.

3. Do not allow the control button to snap back when drawing up a sample. Keep your thumb on the control button allowing it to return gradually.

Part C: How to Dial the Volume

1. Rotate the volume knob to the desired volume. The micropipet can measure only quantities between 1 and 20 ul. Notice however, that there are three number windows. The top window shows 0, 1 and 2 which represent 0, 10, and 20 ul. The middle window shows values from 0 to 9 ul. The bottom window shows 0 to 9 which represent 0.0 to 0.9 ul. Place on the blank lines the measurement indicated by each set of windows below.

0 1

7 = ________ul 3 = ________ul

3 7

2. Seeing what you are doing is very important. So always hold the micropipet in a vertical position at eye level when measuring a liquid.

Part D: Drawing Up a Sample

1. Practice pushing the yellow control knob. You should feel 2 stops. The first stop provides you with the dialed volume on the window. You use this stop when drawing up the desired volume. The second stop will release the desired volume. You push the plunger all the way when releasing the liquid.

2. Push the pipettor firmly into one of the disposable tips in the tip box.

3. Push down on the yellow control button with your thumb to the first stop and hold the button in that position. Dip the pipette tip into the liquid to be withdrawn (never dip into the liquid deeper than the top of the tip) and slowly raise the control button to draw up the liquid into the tip.

Part E: Dispensing the Measured Liquid

1. Insert the tip into a tube and slowly push the control button down to the first stop, wait a second. Now push the control button down to the second stop to blow out the final amount of liquid. Keep the button fully depressed and while lifting the micropipet from the liquid drag the tip along the side of the tube. Now release the control button by slowly raising your thumb. DON'T let the button snap back!

2. Now dispose of the used tip by placing it in the waste container. Hold the micropipet over the waste container and depress the eject button (raised blue area next to the control button) with your index finger. The tip will pop off the end of the pipettor.

Part F: Practice Withdrawing and Dispensing

1. Dial the micropipet to 10.0 ul. Each partner will withdraw three samples from the tube labeled H2O and release them into a second tube following all the steps above except do not eject the tip until you have finished transferring your three samples.

2. Repeat the practice for 12 ul, 5 ul, and 19 ul.

Part G: Loading a Gel

1. In a culture bowl submerged under water is an agar gel with two rows of small rectangular wells (pockets for holding a small amount of liquid). For practice, you and your partner are going to load the wells with ordinary food coloring. See the drawing below to understand how to place the pipette tip into the well. Be sure you don't push the tip into the gel. YES, use both hands and support your elbows on the lab table top.

2. Use the scheme below for loading your gel. Begin by loading the end row of wells then move to the middle row. Load all the red lanes first, green second and finally yellow. You only need to change the tip when you switch to a different color. This saves time and tips.

   Lane #s        Color      Volume              Lane #s     Color         Volume        
      1            Red       8.2 ul                 5        Green         2.5 ul        
      2           Green      6.8 ul                 6        Yellow        7.5 ul        
      3           Yellow     9.3 ul                 7        Red           3.9ul         
      4            Red       4.7 ul                 8        Green         11.2 ul

Dial the micropipet to 15 ul and place this amount of water into an empty microcentrifuge tube. Repeat this step using the same tip. Now dial to 2 ul and add this amount of loading dye into the H₂O tube. Since the loading dye is very dense and heavy, it has a tendency to sink to the bottom of the mixture. To make sure that you have a well-mixed solution, place your micropipet tip into the mixture and stir it around. While stirring, depress the control button to the first stop and release it slowly. Do this several times to thoroughly mix the tube contents. When you feel that the contents are mixed completely, depress the control button to the second stop and remove the micropipet tip from the tube while keeping the control button depressed. Change tips. Allow your partner to repeat the procedure using the same microcentrifuge tube that you used. Be sure your partner adds 30 ml of water and 2 ml of loading dye. Repeat the technique twice. You can add and mix in the same tube. Just be sure to change tips between mixing and adding fresh water and dye. In the future, you should use this stir and pump technique before you remove any liquid sample from one tube to be transferred to another tube.

Part I: Extra Practice

1. Cut a piece of wax paper or parafilm. Lay it flat on the desk.

2. Adjust the micropipet to 12 ul.

3. Using only one food color, write your name in 12 ul drops on the plastic paper.

4. Repeat until the drops are equally spaced and your name can be clearly read.

Analysis

1. How precisely can a digital micropipet measure volume?

2. When should you use a new disposable tip?

3. What is the purpose of the first stop on the control button?

5. What is the purpose of the second stop on the control button?

6. Why should you use the mixing technique before drawing up a sample from a stock solution?